RESUMO
Dictyostelium represents a stripped-down model for understanding how cells make decisions during development. The complete life cycle takes around a day and the fully differentiated structure is composed of only two major cell types. With this apparent reduction in "complexity," single cell transcriptomics has proven to be a valuable tool in defining the features of developmental transitions and cell fate separation events, even providing causal information on how mechanisms of gene expression can feed into cell decision-making. These scientific outputs have been strongly facilitated by the ease of non-disruptive single cell isolation-allowing access to more physiological measures of transcript levels. In addition, the limited number of cell states during development allows the use of more straightforward analysis tools for handling the ensuing large datasets, which provides enhanced confidence in inferences made from the data. In this chapter, we will outline the approaches we have used for handling Dictyostelium single cell transcriptomic data, illustrating how these approaches have contributed to our understanding of cell decision-making during development.
Assuntos
Dictyostelium , Perfilação da Expressão Gênica , Análise de Célula Única , Transcriptoma , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Análise de Célula Única/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. We address this issue using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series identifies a sharp 'jump' in global gene expression marking functionally different cell states. By simultaneously imaging the physiological dynamics of transcription and signalling, we show the jump coincides with the onset of collective oscillations of cAMP. Optogenetic control of cAMP pulses shows that different jump genes respond to distinct dynamic features of signalling. Late jump gene expression changes are almost completely dependent on cAMP, whereas transcript changes at the onset of the jump require additional input. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.
Assuntos
Dictyostelium , Dictyostelium/genética , Transdução de Sinais/genética , Transcriptoma , Perfilação da Expressão GênicaRESUMO
The distribution of mRNA in tissue is determined by the balance between transcription and decay. Understanding the control of RNA decay during development has been somewhat neglected compared with transcriptional control. Here, we explore the potential for mRNA decay to trigger rapid cell state transitions during development, comparing a bistable switch model of cell state conversion with experimental evidence from different developmental systems. We also consider another potential role for large-scale RNA decay that has emerged from studies of stress-induced cell state transitions, in which removal of mRNA unblocks the translation machinery to prioritise the synthesis of proteins that establish the new cell state.
Assuntos
Regulação da Expressão Gênica , RNA , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estabilidade de RNA/genéticaRESUMO
A persistent view of cell fate choices during development entails centralized control by so-called master regulators. A recent single-cell study of the large-scale fate specification during mammalian gastrulation (Mittnenzweig et al., 2021) implies the prevalence of more distributed forms of control.
Assuntos
Gastrulação , Animais , Diferenciação CelularRESUMO
Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing Dictyostelium cells undergo highly efficient dedifferentiation, completed by most cells within 24 hr. We use this rapid response to investigate the control features of dedifferentiation, combining single cell imaging with high temporal resolution transcriptomics. Gene expression during dedifferentiation was predominantly a simple reversal of developmental changes, with expression changes not following this pattern primarily associated with ribosome biogenesis. Mutation of genes induced early in dedifferentiation did not strongly perturb the reversal of development. This apparent robustness may arise from adaptability of cells: the relative temporal ordering of cell and molecular events was not absolute, suggesting cell programmes reach the same end using different mechanisms. In addition, although cells start from different fates, they rapidly converged on a single expression trajectory. These regulatory features may contribute to dedifferentiation responses during regeneration.
Assuntos
Desdiferenciação Celular/genética , Dictyostelium/citologia , Expressão Gênica , Mutação , Dictyostelium/fisiologia , Perfilação da Expressão Gênica , Análise de Célula Única , Fatores de TranscriçãoRESUMO
Stimulation of the ERK/MAPK pathway is required for the exit from pluripotency and onset of differentiation in mouse embryonic stem cells (ESCs). The dynamic behaviour of ERK activity in individual cells during this transition is unclear. Using a FRET-based biosensor, we monitored ERK signalling dynamics of single mouse ESCs during differentiation. ERK activity was highly heterogeneous, with considerable variability in ERK signalling between single cells within ESC colonies. Different triggers of differentiation induced distinct ERK activity profiles. Surprisingly, the dynamic features of ERK signalling were not strongly coupled to loss of pluripotency marker expression, regardless of the differentiation stimulus, suggesting the normal dynamic range of ERK signalling is not rate-limiting in single cells during differentiation. ERK signalling dynamics were sensitive to the degree of cell crowding and were similar in neighbouring cells. Sister cells from a mitotic division also showed more similar ERK activity, an effect that was apparent whether cells remained adjacent or moved apart after division. These data suggest a combination of cell lineage and niche contributes to the absolute level of ERK signalling in mouse ESCs.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Transdução de Sinais , Animais , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Transferência Ressonante de Energia de Fluorescência , Marcadores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Mitose , Proteína Homeobox Nanog/metabolismoRESUMO
Embryonic development involves extensive and often rapid cell proliferation. An unavoidable side effect of cell proliferation is DNA damage. The consequences of spontaneous DNA damage during development are not clear. Here, we define an approach to determine the effects of DNA damage on cell fate choice. Using single cell transcriptomics, we identified a subpopulation of Dictyostelium cells experiencing spontaneous DNA damage. Damaged cells displayed high expression of rad51, with the gene induced by multiple types of genotoxic stress. Using live imaging, we tracked high Rad51 cells from differentiation onset until cell fate assignment. High Rad51 cells were shed from multicellular structures, excluding damaged cells from the spore population. Cell shedding resulted from impaired cell motility and defective cell-cell adhesion, with damaged cells additionally defective in activation of spore gene expression. These data indicate DNA damage is not insulated from other aspects of cell physiology during development and multiple features of damaged cells prevent propagation of genetic error. Our approach is generally applicable for monitoring rare subpopulations during development, and permits analysis of developmental perturbations occurring within a physiological dynamic range.
Assuntos
Dano ao DNA , Dictyostelium/fisiologia , Regulação da Expressão Gênica , Adesão Celular , Linhagem da Célula , Movimento Celular , Fenômenos Fisiológicos Celulares , Reparo do DNA , Ligação Proteica , Rad51 Recombinase/metabolismo , TranscriptomaRESUMO
The generation of multiple fates from a uniform cell population via self-organisation is a recurring feature in development and regeneration. However, for most self-organising systems, we have little understanding of the processes that allow cells to become different. One of the clearest examples of developmental self-organisation is shown by Dictyostelium, with cells segregating into two major fates, stalk and spore, within multicellular aggregates. To characterise the gene expression decisions that underlie this cell fate bifurcation, we carried out single cell transcriptomics on Dictyostelium aggregates. Our data show the transition of progenitors into prespore and prestalk cells occurs via distinct developmental intermediates. Few cells were captured switching between states, with minimal overlap in fate marker expression between cell types, suggesting states are discrete and transitions rapid. Surprisingly, fate-specific transcript dynamics were a small proportion of overall gene expression changes, with transcript divergence coinciding precisely with large-scale remodelling of the transcriptome shared by prestalk and prespore cells. These observations suggest the stepwise separation of cell identity is temporally coupled to global expression transitions common to both fates.
Assuntos
Linhagem da Célula , Dictyostelium/genética , Dictyostelium/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Processos Estocásticos , Ciclo Celular , Linhagem Celular , Hibridização in Situ Fluorescente , Análise de Componente Principal , RNA-Seq , Análise de Célula Única/métodos , TranscriptomaRESUMO
Gene expression levels vary greatly within similar cells, even within clonal cell populations [1]. These spontaneous expression differences underlie cell fate diversity in both differentiation and disease [2]. The mechanisms responsible for generating expression variability are poorly understood. Using single-cell transcriptomics, we show that transcript variability emerging during Dictyostelium differentiation is driven predominantly by repression rather than activation. The increased variability of repressed genes was observed over a broad range of expression levels, indicating that variability is actively imposed and not a passive statistical effect of the reduced numbers of molecules accompanying repression. These findings can be explained by a simple model of transcript production, with expression controlled by the frequency, rather than the magnitude, of transcriptional firing events. Our study reveals that the generation of differences between cells can be a direct consequence of the basic mechanisms of transcriptional regulation.
Assuntos
Dictyostelium/genética , Regulação da Expressão Gênica , Transcrição Gênica , Análise de Célula ÚnicaRESUMO
Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Sophisticated tools used to investigate their activity in living cells include probes based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation, and photoactivation. However, such methods are of limited use in quickly migrating cells due to a short time available for image acquisition leading to a low signal-to-noise ratio. Attempts to remedy this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell photosensitivity. Here we present design and characterization of a new fluorescent probe that selectively binds to active form of Rac1 GTPases, and demonstrate its superior properties for imaging in highly motile Dictyostelium cells. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase and was selected on the basis of yeast two-hybrid screen, GST pull-down assay and FRET measurements by fluorescence lifetime imaging microscopy. DPAKa(GBD) probe binds specifically to GTP-bound Rac1 at the cell membrane and features a low cytoplasmic background. The main advantage of DPAKa(GBD) in comparison with similar probes is its finely graded intensity distribution along the entire plasma membrane, which enables quantitative measurements of the Rac1 activity in different parts of the membrane. Finally, expression of DPAKa(GBD) induces no adverse effects on cell growth, motility and cytokinesis.
Assuntos
Dictyostelium/metabolismo , Transferência Ressonante de Energia de Fluorescência , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células Cultivadas , Dictyostelium/citologia , Proteínas rac1 de Ligação ao GTP/análiseRESUMO
We describe a simple optical configuration for dark-field microscopy at low magnification, realized with the use of standard microscope components. An inherent high contrast makes this method attractive for computer-assisted tracking and counting of microorganisms. We applied this setup for dark-field microscopy to measure the speed of migrating Dictyostelium amoebae.